Endothelial Cell Network Formation Research Articles

SEMA3G regulates BMP9 inhibition of VEGF-mediated migration and network formation in pulmonary endothelial cells

AimsBone morphogenetic protein-9 (BMP9) is critical for bone morphogenetic protein receptor type-2 (BMPR2) signalling in pulmonary vascular endothelial cells. Furthermore, human genetics studies support the central role of disrupted BMPR2 mediated BMP9 signalling in vascular endothelial cells in the initiation of pulmonary arterial hypertension (PAH). In addition, loss-of-function mutations in BMP9 have been identified in PAH patients. BMP9 is considered to play an important role in vascular homeostasis and quiescence. Methods and resultsWe identified a novel BMP9 target as the class-3 semaphorin, SEMA3G. Although originally identified as playing a role in neuronal development, class-3 semaphorins may have important roles in endothelial function. Here we show that BMP9 transcriptional regulation of SEMA3G occurs via ALK1 and the canonical Smad pathway, requiring both Smad1 and Smad5. Knockdown studies demonstrated redundancy between type-2 receptors in that BMPR2 and ACTR2A were compensatory. Increased SEMA3G expression by BMP9 was found to be regulated by the transcription factor, SOX17. Moreover, we observed that SEMA3G regulates VEGF signalling by inhibiting VEGFR2 phosphorylation and that VEGF, in contrast to BMP9, negatively regulated SEMA3G transcription. Functional endothelial cell assays of VEGF-mediated migration and network formation revealed that BMP9 inhibition of VEGF was abrogated by SEMA3G knockdown. Conversely, treatment with recombinant SEMA3G partially mimicked the inhibitory action of BMP9 in these assays. ConclusionsThis study provides further evidence for the anti-angiogenic role of BMP9 in microvascular endothelial cells and these functions are mediated at least in part via SOX17 and SEMA3G induction.

Biomimetic management of bone healing stages: MOFs induce tunable degradability and enhanced angiogenesis-osteogenesis coupling

Currently, biodegradable biomaterials are extensively used in bone regeneration. Nevertheless, a significant challenge remains in accurately adjusting their degradation rates to match bone healing stages to achieve bone regeneration. Herein, we present a novel biodegradable bone grafts integrated with zeolite imidazole framework-8 (ZIF-8) nanocrystals, fabricated via microinjection molding. Apart from the reinforcing effect derived from the in situ fibrillation of PCL in PLGA, nanoscale ZIF-8 is inspiringly demonstrated to control the PLGA degradation. Both molecular dynamic simulation and experimental results indicate that Zn2+ derived from ZIF-8 nanocrystals acts as a Lewis acid to participate the degradation of PLGA matrix, thus successfully reconciles the contradiction between degradation of matrix and maintenance of reinforcing phase. Moreover, nanometric ZIF-8 modified bone grafts improve the angiogenesis-osteogenesis coupling. In vitro, it directly boosts HIF-1α and capillary-like network formation of endothelial cells (ECs), and RUNX2 and OCN expression in bone marrow mesenchymal stem cells (BMSCs). In vivo, bone grafts with a moderate concentration (0.5 %) of ZIF-8 nanoparticles aid vascularized bone regeneration by tunable biodegradation matching bone healing stages. Further, RNA sequencing shows PLGA/PCL@nZ bone grafts promote angiogenesis-osteogenesis by cooperatively regulating calcium signaling pathway in both ECs and BMSCs. Collectively, this study delineates a groundbreaking bone repair strategy with profound clinical application potential.

Phototuning of Hyaluronic-Acid-Based Hydrogel Properties to Control Network Formation in Human Vascular Endothelial Cells.

In vitro network formation by endothelial cells serves as a fundamental model for studies aimed at understanding angiogenesis. The morphogenesis of these cells to form a network is intricately regulated by the mechanical and biochemical properties of the extracellular matrix. Here the effects of modulating these properties in hydrogels derived from phenolated hyaluronic acid (HA-Ph) and phenolated gelatin (Gelatin-Ph) are presented. Visible-light irradiation in the presence of tris(2,2'-bipyridyl)ruthenium(II) chloride hexahydrate and sodium persulfate induces the crosslinking of these polymers, thereby forming a hydrogel and degrading HA-Ph. Human vascular endothelial cells form networks on the hydrogel prepared by visible-light irradiation for 45min (42W cm-2 at 450nm) but not on the hydrogels prepared by irradiation for 15, 30, or 60min. The irradiation time-dependent degradation of HA-Ph and the changes in the mechanical stiffness of the hydrogels, coupled with the expressions of RhoA and β-actin genes and CD44 receptors in the cells, reveal that the network formation is synergistically influenced by the hydrogel stiffness and HA-Ph degradation. These findings highlight the potential of tailoring HA-based hydrogel properties to modulate human vascular endothelial cell responses, which is critical for advancing their application in vascular tissue engineering.

Effects of a Bioengineered Allogeneic Cellularized Construct (BACC) on Primary Human Macrophage Phenotype.

The mechanisms behind the pro-healing effects of multicellular, bioengineered allogeneic cellularized constructs (BACC) are not known. Macrophages are key regulators of every phase of the wound healing process and the primary cells that mediate the response to biomaterials. It is hypothesized that cells within the BACC modulate macrophage behavior, which may contribute to the mechanism by which BACC promotes healing. To probe the influence of cells within the BACC compared to effects of the underlying collagen substrate, primary human macrophages are cultured in direct or indirect contact with BACC or with the same collagen substrate used in the BACC manufacturing. Macrophage phenotype is characterized over time via multiplex gene expression, protein secretion, multidimensional flow cytometry, and functional assays with fibroblasts and endothelial cells. The BACC causes macrophages to exhibit a predominately reparative phenotype over time compared to relevant collagen substrate controls, with multiple subpopulations expressing both pro-inflammatory and reparative markers. Conditioned media from macrophage-BACC co-cultures causes distinct effects on fibroblast and endothelial cell proliferation, migration, and network formation. Given the critical role of the reparative macrophage phenotype in wound healing, these results suggest that modulation of macrophage phenotype may be a critical part of the mechanisms behind BACC's pro-healing effects.

Targeting the microenvironment in the treatment of arteriovenous malformations

Extracranial arteriovenous malformations (AVMs) are regarded as rare diseases and are prone to complications such as pain, bleeding, relentless growth, and high volume of shunted blood. Due to the high vascular pressure endothelial cells of AVMs are exposed to mechanical stress. To control symptoms and lesion growth pharmacological treatment strategies are urgently needed in addition to surgery and interventional radiology. AVM cells were isolated from three patients and exposed to cyclic mechanical stretching for 24 h. Thalidomide and bevacizumab, both VEGF inhibitors, were tested for their ability to prevent the formation of circular networks and proliferation of CD31+ endothelial AVM cells. Furthermore, the effect of thalidomide and bevacizumab on stretched endothelial AVM cells was evaluated. In response to mechanical stress, VEGF gene and protein expression increased in patient AVM endothelial cells. Thalidomide and bevacizumab reduced endothelial AVM cell proliferation. Bevacizumab inhibited circular network formation of endothelial AVM cells and lowered VEGF gene and protein expression, even though the cells were exposed to mechanical stress. With promising in vitro results, bevacizumab was used to treat three patients with unresectable AVMs or to prevent regrowth after incomplete resection. Bevacizumab controlled bleeding, pulsation, and pain over the follow up of eight months with no patient-reported side effects. Overall, mechanical stress increases VEGF expression in the microenvironment of AVM cells. The monoclonal VEGF antibody bevacizumab alleviates this effect, prevents circular network formation and proliferation of AVM endothelial cells in vitro. The clinical application of bevacizumab in AVM treatment demonstrates effective symptom control with no side effects.Graphical abstractMechanical stress increases VEGF expression in endothelial AVM cells, possibly causing the VEGF upregulation in the microenvironment of AVM cells. The resulting RAS/RAF/MEK/ERK signaling in leads to progression of fast-flow malformations. The monoclonal VEGF-A antibody bevacizumab alleviates this effect, prevents circular network formation and proliferation of AVM endothelial cells in vitro. Sporadically occurring slow-flow malformations (LMs, VMs) have mutations in TEK or PIK3CA. TEK encodes the endothelial receptor tyrosine kinase TIE2. Sporadic extracranial fast-flow malformations (AVMs) show mutations in KRAS, BRAF and MAP2K1, which encodes the dual specificity mitogen-activated protein kinase MEK1. Combined targeting of the molecular causes of the disease could be key to achieve symptom control and reduce lesion growth. Orange: gain-of-function; Blue, circled with orange: enhanced signaling.

GATA6 regulates anti-angiogenic properties in human cardiac fibroblasts via modulating LYPD1 expression.

Fibroblasts contribute to the structure and function of tissue and organs; however, their properties differ in each organ given the topographic variation in gene expression among tissues. We previously reported that LYPD1, which is expressed in cardiac fibroblasts, has the capacity to inhibit sprouting of vascular endothelial cells. LYPD1 has been shown to be highly expressed in the human brain and heart, but the regulation of LYPD1 expression in cardiac fibroblasts has not been elucidated in detail. To identify the LYPD1-modulating transcription factor, motif enrichment analysis and differential expressed gene analysis using microarray data were performed. Quantitative real-time PCR was used to evaluate gene expression. Gene silencing were performed by transfection of siRNA. Western blot analyzed protein expression in NHCF-a. To assess the effect of GATA6 on the regulation of LYPD1 gene expression, dual-luciferase reporter assay was performed. Co-culture and rescue experiments were performed to evaluate endothelial network formation. Motif enrichment analysis and differential expressed gene analysis using microarray data and quantitative real-time PCR revealed that CUX1, GATA6, and MAFK were candidate transcription factors. Of these, the inhibition of GATA6 expression using siRNA decreased LYPD1 gene expression and co-expression of GATA6 with a reporter vector containing the upstream sequence of the LYPD1 gene resulted in increased reporter activity. Endothelial cell network formation was attenuated when co-cultured with cardiac fibroblasts, but it was significantly restored when co-cultured with cardiac fibroblasts wherein the expression of GATA6 was knocked down with siRNA. GATA6 regulate the anti-angiogenic properties of cardiac fibroblasts by modulating LYPD1 expression.

Spatial-Selective Volumetric 4D Printing and Single-Photon Grafting of Biomolecules within Centimeter-Scale Hydrogels via Tomographic Manufacturing.

Conventional additive manufacturing and biofabrication techniques are unable to edit the chemicophysical properties of the printed object postprinting. Herein, a new approach is presented, leveraging light-based volumetric printing as a tool to spatially pattern any biomolecule of interest in custom-designed geometries even across large, centimeter-scale hydrogels. As biomaterial platform, a gelatin norbornene resin is developed with tunable mechanical properties suitable for tissue engineering applications. The resin can be volumetrically printed within seconds at high resolution (23.68 ± 10.75 μm). Thiol-ene click chemistry allows on-demand photografting of thiolated compounds postprinting, from small to large (bio)molecules (e.g., fluorescent dyes or growth factors). These molecules are covalently attached into printed structures using volumetric light projections, forming 3D geometries with high spatiotemporal control and ≈50 μm resolution. As a proof of concept, vascular endothelial growth factor is locally photografted into a bioprinted construct and demonstrated region-dependent enhanced adhesion and network formation of endothelial cells. This technology paves the way toward the precise spatiotemporal biofunctionalization and modification of the chemical composition of (bio)printed constructs to better guide cell behavior, build bioactive cue gradients. Moreover, it opens future possibilities for 4D printing to mimic the dynamic changes in morphogen presentation natively experienced in biological tissues.

Abstract 4597: TIMP1 is a major contributor of the angiogenic priming of tumor associated fibroblasts in lung adenocarcinoma

Abstract Background: Lung adenocarcinoma (ADC) and squamous cell carcinoma (SCC) are the most common histologic subtypes in lung cancer. Intriguingly, ADC and SCC exhibit distinct responses to the multi-tyrosine kinase inhibitor nintedanib and other antiangiogenic drugs, suggesting that angiogenesis may depend on the histologic subtype in lung cancer. In addition, tumor-associated fibroblasts (TAFs) are known regulators of angiogenesis, and we have recently reported that TAFs exhibit enhanced fibrosis and response to nintedanib in ADC compared to SCC, owing to the stronger epigenetic repression of SMAD3 in SCC-TAFs caused by excessive exposure to cigarette smoke particles. However, how the histotype-dependent fibrotic phenotype in TAFs impacts their pro-angiogenic functions remains unknown. Methods: A panel of angiogenesis markers was assessed in patient samples using publicly available databases. The pro-angiogenic function of the conditioned medium of TAFs from ADC and SCC patients pre-activated with TGF-β1 was analyzed in vitro using migration and network formation assays of endothelial cells. The secretion of pro-angiogenic factors in TGF-β1-activated TAFs was analyzed using an angiogenesis antibody blot array. Selected factors were functionally validated in vitro and in vivo using genetic models. Results: All angiogenesis markers were consistently upregulated in ADC compared to SCC concomitantly with a lower necrosis in ADC, unveiling a larger angiogenesis in ADC. We also observed that the conditioned medium of TGF-β1-activated TAFs elicited a larger endothelial cell network formation and migration in ADC than SCC, revealing that ADC-TAFs exhibit enhanced angiogenesis. Our immunoblot array analysis identified a subset of pro-angiogenic factors that were selectively overexpressed in ADC-TAFs compared to SCC-TAFs, including TIMP-1. Notably, TIMP-1 overexpression in ADC-TAFs was SMAD3-dependent and knocking down TIMP-1 by siRNA in ADC-TAFs impaired angiogenesis in vitro and in tumor xenografts in vivo. Conclusions: Our results reveal a larger angiogenesis in ADC compared to SCC, and implicate the TGF-β1/SMAD3/TIMP-1 pathway in the enhanced angiogenesis of ADC-TAFs. Our findings identify a biological process underlying the poor response of SCC to antiangiogenic therapies like nintedanib, and provide a rationale for the earlier metastasis of ADC patients compared to SCC patients based on the enhanced pro-angiogenic role of ADC-TAFs. Citation Format: Natalia Isabel Diaz Valdivia, Paula Duch, Rafael Ikemori, Eduard Monso, Noemi Reguart, Jordi Alcaraz. TIMP1 is a major contributor of the angiogenic priming of tumor associated fibroblasts in lung adenocarcinoma. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4597.

Skeletonema marinoi Extracts and Associated Carotenoid Fucoxanthin Downregulate Pro-Angiogenic Mediators on Prostate Cancer and Endothelial Cells.

The exploration of natural preventive molecules for nutraceutical and pharmaceutical use has recently increased. In this scenario, marine microorganisms represent an underestimated source of bioactive products endowed with beneficial effects on health that include anti-oxidant, anti-inflammatory, differentiating, anti-tumor, and anti-angiogenic activities. Here, we tested the potential chemopreventive and anti-angiogenic activities of an extract from the marine coastal diatom Skeletonema marinoi Sarno and Zingone (Sm) on prostate cancer (PCa) and endothelial cells. We also tested one of the main carotenoids of the diatom, the xanthophyll pigment fucoxanthin (Fuco). Fuco from the literature is a potential candidate compound involved in chemopreventive activities. Sm extract and Fuco were able to inhibit PCa cell growth and hinder vascular network formation of endothelial cells. The reduced number of cells was partially due to growth inhibition and apoptosis. We studied the molecular targets by qPCR and membrane antibody arrays. Angiogenesis and inflammation molecules were modulated. In particular, Fuco downregulated the expression of Angiopoietin 2, CXCL5, TGFβ, IL6, STAT3, MMP1, TIMP1 and TIMP2 in both prostate and endothelial cells. Our study confirmed microalgae-derived drugs as potentially relevant sources of novel nutraceuticals, providing candidates for potential dietary or dietary supplement intervention in cancer prevention approaches.

Nailfold Videocapillaroscopy for Non-Invasive Assessment of Microcirculation and Prognostic Correlation with Endothelial Dysfunction, Cardiovascular Risk Factors, and Non-HLA Antibodies in Heart Transplant Recipients: A Pilot Study.

Early identification of allograft vasculopathy and the concomitant elimination of adverse risk factors is essential for improving the long-term prognosis of heart transplant (HTx) recipients with underlying cardiovascular disease (CVD). The major aim of this pilot study was to conduct a non-invasive imaging evaluation of the HTx patient microcirculation by employing nailfold video-capillaroscopy (NVC) in a well-characterized patient and control cohort, and to correlate these data with endothelial cell function, accompanied by studies of traditional cardiovascular risk factors and non-HLA antibodies in HTx recipients. Ten patients undergoing HTx (mean age of 38 ± 14 years) were recruited for the study and compared to a control group of 12 well-matched healthy volunteers (mean age 35 ± 5 years) with normal body mass index (BMI). Detailed medical records were collected from all individuals. NVC was performed using CapillaryScope 200 MEDL4N microscope. For functional readout and correlation analysis, endothelial cell network formation in conjunction with measurements of patient serum levels of vascular endothelial growth factor (VEGF) and non-HLA autoantibodies directed against the angiotensin II type-1-receptor (anti-AT1R-Ab), endothelin-1 type-A-receptor (anti-ETAR-Ab), protease-activated receptor-1 (anti-PAR-1-Ab), and VEGF-A (anti-VEGF-A-Ab) were studied. Our NVC analysis found that the average apical loop diameter of nailfold capillaries was significantly increased in HTx recipients (p = 0.001). In addition, HTx patients with more prominent changes in capillaroscopic patterns were characterized by the presence of traditional cardiovascular risk factors, and HTx patients had increased levels of anti-AT1R-ab, anti-ETAR-ab, and anti-VEGF-A-Ab (p = 0.017, p = 0.025, and p = 0.003, respectively). Capillary diameters most strongly correlated with elevated serum levels of troponin T and triglycerides (R = 0.69, p = 0.028 and R = 0.81, p = 0.004, respectively). In conclusion, we found that an abnormal NVC pattern in HTx patients is associated with traditional CVD risk factors and that NVC is a useful non-invasive tool to conveniently monitor changes in the microvasculature of HTx patients.

Integrin binding peptides facilitate growth and interconnected vascular-like network formation of rat primary cortical vascular endothelial cells in vitro.

Neovascularization and angiogenesis in the brain are important physiological processes for normal brain development and repair/regeneration following insults. Integrins are cell surface adhesion receptors mediating important function of cells such as survival, growth and development during tissue organization, differentiation and organogenesis. In this study, we used an integrin-binding array platform to identify the important types of integrins and their binding peptides that facilitate adhesion, growth, development, and vascular-like network formation of rat primary brain microvascular endothelial cells. Brain microvascular endothelial cells were isolated from rat brain on post-natal day 7. Cells were cultured in a custom-designed integrin array system containing short synthetic peptides binding to 16 types of integrins commonly expressed on cells in vertebrates. After 7 days of culture, the brain microvascular endothelial cells were processed for immunostaining with markers for endothelial cells including von Willibrand factor and platelet endothelial cell adhesion molecule. 5-Bromo-2'-dexoyuridine was added to the culture at 48 hours prior to fixation to assess cell proliferation. Among 16 integrins tested, we found that α5β1, αvβ5 and αvβ8 greatly promoted proliferation of endothelial cells in culture. To investigate the effect of integrin-binding peptides in promoting neovascularization and angiogenesis, the binding peptides to the above three types of integrins were immobilized to our custom-designed hydrogel in three-dimensional (3D) culture of brain microvascular endothelial cells with the addition of vascular endothelial growth factor. Following a 7-day 3D culture, the culture was fixed and processed for double labeling of phalloidin with von Willibrand factor or platelet endothelial cell adhesion molecule and assessed under confocal microscopy. In the 3D culture in hydrogels conjugated with the integrin-binding peptide, brain microvascular endothelial cells formed interconnected vascular-like network with clearly discernable lumens, which is reminiscent of brain microvascular network in vivo. With the novel integrin-binding array system, we identified the specific types of integrins on brain microvascular endothelial cells that mediate cell adhesion and growth followed by functionalizing a 3D hydrogel culture system using the binding peptides that specifically bind to the identified integrins, leading to robust growth and lumenized microvascular-like network formation of brain microvascular endothelial cells in 3D culture. This technology can be used for in vitro and in vivo vascularization of transplants or brain lesions to promote brain tissue regeneration following neurological insults.

Human Umbilical Vein Endothelial Cells Form a Network on a Hyaluronic Acid/Gelatin Composite Hydrogel Moderately Crosslinked and Degraded by Hydrogen Peroxide.

The study of the capillary-like network formation of human umbilical vein endothelial cells (HUVECs) in vitro is important for understanding the factors that promote or inhibit angiogenesis. Here, we report the behavior of HUVECs on the composite hydrogels containing hyaluronic acid (HA) and gelatin with different degrees of degradation, inducing the different physicochemical properties of the hydrogels. The hydrogels were obtained through horseradish peroxidase (HRP)-catalyzed hydrogelation consuming hydrogen peroxide (H2O2, 16 ppm) supplied from the air, and the degradation degree was tuned by altering the exposure time to the air. The HUVECs on the composite hydrogel with intermediate stiffness (1.2 kPa) obtained through 120 min of the exposure were more elongated than those on the soft (0.4 kPa) and the stiff (2.4 kPa) composite hydrogels obtained through 15 min and 60 min of the exposure, respectively. In addition, HUVECs formed a capillary-like network only on the stiff composite hydrogel although those on the hydrogels with comparable stiffness but containing gelatin alone or alginate instead of HA did not form the network. These results show that the HA/gelatin composite hydrogels obtained through the H2O2-mediated crosslinking and degradation could be a tool for studies using HUVECs to understand the promotion and inhibition of angiogenesis.

Preclinical anti‐angiogenic and anti‐cancer activities of BAY1143269 in glioblastoma via targeting oncogenic protein expression

Glioblastoma angiogenesis is critical for tumor growth, making it an appealing target for treatment development. BAY1143269 is a novel inhibitor of mitogen‐activated protein kinase interacting serine/threonine‐protein kinase 1 (MKN1) and has potent anti‐cancer activity. We identified BAY1143269 as an angiogenesis inhibitor, by in vitro and in vivo glioblastoma angiogenesis models. BAY1143269 inhibited the capillary network formation of glioblastoma microvascular endothelial cells (GMECs), particularly the early stage of tubular structure formation. It also inhibited migration and proliferation, and induced apoptosis of GMECs isolated from glioblastoma patients. We found that BAY1143269 acted on GMECs by suppressing the eukaryotic translation initiation factor 4E (eIF4E) and eIF4E‐mediated expression of oncogenic proteins, including those involved in cell cycle, epithelial‐mesenchymal transition (EMT), and pro‐survival. In addition, BAY1143269 suppressed eIF4E phosphorylation, inhibited proliferation, and induced apoptosis of glioblastoma cells. Interestingly, it reduced vascular endothelial growth factor (VEGF) level in tumor cells and culturing medium, demonstrating the inhibitory effect of BAY1143269 on tumor proangiogenic microenvironment. We finally challenged BAY1143269 on the glioblastoma xenograft mice model and observed a significant tumor growth reduction without toxicity in mice receiving oral BAY1143269. Immunoblotting analysis demonstrated significantly less phosphorylated‐eIF4E (p‐eIF4E), cluster of differentiation 31 (CD31) (microvascular endothelial cell marker), and VEGF in tumors from drug‐treated mice. In summary, the inhibition of glioblastoma angiogenesis with BAY1143269 may provide an alternative approach for anti‐glioblastoma therapy.

Context dependent role of p53 during the interaction of hepatocellular carcinoma and endothelial cells.

During the progression of hepatocellular carcinoma (HCC), several angiogenic factors are overexpressed in the hepatic microenvironment, which play a critical role in governing the phenotype of the endothelial cells. Mutation in the p53 gene (TP53) is a common event in HCC that may dysregulate the angiogenic signals. However, their functional messages remain largely unexplored at the onset of metastasis. Role of p53 was studied by siRNA mediated silencing of p53 in HepG2 cells (WTp53), collecting and analyzing their conditioned medium, followed by indirect co-culture with endothelial cells (HUVECs). Gene and protein expression in HCC cells and endothelial cells was studied by RT-qPCR and western blotting respectively. β-catenin protein expression and localization were analyzed by immunocytochemistry. We have studied a cell-to-cell interaction model to investigate the crosstalk of endothelial and hepatoma cells by either knocking down p53 or by using p53 null low metastatic HCC cell line. In the absence of p53, the HCC cells influence the migration and vascular network formation of endothelial cells through paracrine signaling of VEGF. Secretory VEGF activated the VEGF receptor-2 along with the survival signaling in endothelial cells. However, the β-catenin signal is upregulated in endothelial cells only during interaction with metastatic set up irrespective of absence and presence of p53, indicating context-dependent participation of p53 during communication between hepatoma cells and endothelial cells. This study highlights that the role of p53 on cellular responses during interaction of hepatocellular carcinoma and endothelial cells is distinct to cell types and context.

Synergistic effect of glucagon-like peptide-1 analogue liraglutide and ZnO on the antibacterial, hemostatic, and wound healing properties of nanofibrous dressings

Bacterial infections and poor vascularization delay wound healing, thus necessitating alternative strategies for functional wound dressings. Zinc oxide (ZnO) has been shown to exert a potent antibacterial effect against bacterial species. Similarly, Glucagon-like peptide-1 (GLP-1) analogue liraglutide (LG) has been shown to promote vascularization and improve wound healing. The objective of this research was to investigate the synergistic effect of ZnO nanoparticles (ZnO-NPs) and LG to simultaneously induce antibacterial, hemostatic, and vascularization effects for infected wound healing. Electrospun poly (l-lactide-co-glycolide)/gelatin (PLGA/Gel) membranes containing ZnO-NPs and LG displayed good biocompatibility and hemostatic ability. Both, ZnO-NPs and LG exhibited synergistic antibacterial effect against Staphylococcus aureus and Escherichia coli as well as improved the migration and tubule-like network formation of human umbilical vein endothelial cells (HUVECs) invitro. Once evaluated in a bacterial-infected wound model in rats, the membranes loaded with ZnO-NPs and LG effectively promoted wound healing causing significant reduction in wound area and scar-like tissue formation. Therefore, ZnO-NPs/LG synergism may offer an invaluable solution for the treatment of poorly healing infected wounds.

Angiogenic role of lung pericytes in experimental viral lung injury

Lung pericytes are perivascular stromal cells that surround microvascular endothelial cells. While their roles in fibrogenesis have been explored, their angiogenic functions in experimental lung injury and repair have not been characterized. RNAseq analysis of primary lung pericytes activated ex vivoreveals upregulation of novel angiogenic pathways. One gene of interest we identified is angiopoietin‐L4 (Angptl4). Stromal cells, particularly pericytes, are the primary sources of angiopoietin‐L4 in the lung. ANGPTL4 has been shown to regulate angiogenesis in cancer biology and retinal diseases. We have shown that lung pericyte‐derived ANGPTL4 promotes network formation and monolayer permeability in endothelial cells in vitro. We hypothesize that knockdown of ANGPTL4 in vivo is protective in acute viral lung injury by promoting endothelial integrity and decreasing microvascular leak during injury and resolution. Transgenic mice with inducible genetic deletion of Angptl4 and littermate controls were inoculated with mouse‐adapted human influenza virus (A/PR/8). Mice lacking Angptl4 showed decreased measures of lung injury and protein leak during the acute and resolution phases of lung injury. Future directions include identification of ANGPTL4 signaling pathways and cellular targets in the lung, and characterization of the mechanisms by which pericyte‐derived ANGPTL4 leads to persistence of endothelial dysfunction or activation in viral lung injury.

Capillary morphogenesis gene 2 (CMG2) mediates growth factor-induced angiogenesis by regulating endothelial cell chemotaxis.

Anthrax protective antigen (PA) is a potent inhibitor of pathological angiogenesis with an unknown mechanism. In anthrax intoxication, PA interacts with capillary morphogenesis gene 2 (CMG2) and tumor endothelial marker 8 (TEM8). Here, we show that CMG2 mediates the antiangiogenic effects of PA and is required for growth-factor-induced chemotaxis. Using specific inhibitors of CMG2 and TEM8 interaction with natural ligand, as well as mice with the CMG2 or TEM8 transmembrane and intracellular domains disrupted, we demonstrate that inhibiting CMG2, but not TEM8 reduces growth-factor-induced angiogenesis in the cornea. Furthermore, the antiangiogenic effect of PA was abolished when the CMG2, but not the TEM8, gene was disrupted. Binding experiments demonstrated a broad ligand specificity for CMG2 among extracellular matrix (ECM) proteins. Ex vivo experiments demonstrated that CMG2 (but not TEM8) is required for PA activity in human dermal microvascular endothelial cell (HMVEC-d) network formation assays. Remarkably, blocking CMG2-ligand binding with PA or CRISPR knockout abolishes endothelial cell chemotaxis but not chemokinesis in microfluidic migration assays. These effects are phenocopied by Rho inhibition. Because CMG2 mediates the chemotactic response of endothelial cells to peptide growth factors in an ECM-dependent fashion, CMG2 is well-placed to integrate growth factor and ECM signals. Thus, CMG2 targeting is a novel way to inhibit angiogenesis.

Examination of the Role of Galectins and Galectin Inhibitors in Endothelial Cell Biology.

The growth of new blood vessels is a key event in many (patho) physiological processes, including embryogenesis, wound healing, inflammatory diseases, and cancer. Neovascularization requires different, well-coordinated actions of endothelial cells, i.e., the cells lining the luminal side of all blood vessels. Galectins are involved in several of these activities. In this chapter, we describe methods to study galectins in three key functions of endothelial cells during angiogenesis, i.e., endothelial cell migration, endothelial cell sprouting, and endothelial cell network formation.

The SARS-CoV-2 Spike protein disrupts human cardiac pericytes function through CD147 receptor-mediated signalling: a potential non-infective mechanism of COVID-19 microvascular disease.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes a broad range of clinical responses including prominent microvascular damage. The capacity of SARS-CoV-2 to infect vascular cells is still debated. Additionally, the SARS-CoV-2 Spike (S) protein may act as a ligand to induce non-infective cellular stress. We tested this hypothesis in pericytes (PCs), which are reportedly reduced in the heart of patients with severe coronavirus disease-2019 (COVID-19). Here we newly show that the in vitro exposure of primary human cardiac PCs to the SARS-CoV-2 wildtype strain or the α and δ variants caused rare infection events. Exposure to the recombinant S protein alone elicited signalling and functional alterations, including: (1) increased migration, (2) reduced ability to support endothelial cell (EC) network formation on Matrigel, (3) secretion of pro-inflammatory molecules typically involved in the cytokine storm, and (4) production of pro-apoptotic factors causing EC death. Next, adopting a blocking strategy against the S protein receptors angiotensin-converting enzyme 2 (ACE2) and CD147, we discovered that the S protein stimulates the phosphorylation/activation of the extracellular signal-regulated kinase 1/2 (ERK1/2) through the CD147 receptor, but not ACE2, in PCs. The neutralisation of CD147, either using a blocking antibody or mRNA silencing, reduced ERK1/2 activation, and rescued PC function in the presence of the S protein. Immunoreactive S protein was detected in the peripheral blood of infected patients. In conclusion, our findings suggest that the S protein may prompt PC dysfunction, potentially contributing to microvascular injury. This mechanism may have clinical and therapeutic implications.

Serine 26 in Early Growth Response-1 Is Critical for Endothelial Proliferation, Migration, and Network Formation.

BackgroundVascular endothelial cell proliferation, migration, and network formation are key proangiogenic processes involving the prototypic immediate early gene product, Egr‐1 (early growth response‐1). Egr‐1 undergoes phosphorylation at a conserved Ser26 but its function is completely unknown in endothelial cells or any other cell type.Methods and ResultsA CRISPR/Cas9 strategy was used to introduce a homozygous Ser26>Ala mutation into endogenous Egr‐1 in human microvascular endothelial cells. In the course of generating mutant cells, we produced cells with homozygous deletion in Egr ‐1 caused by frameshift and premature termination. We found that Ser26 mutation in Egr‐1, or Egr‐1 deletion, perturbed endothelial cell proliferation in models of cell counting or real‐time growth using the xCELLigence System. We found that Ser26 mutation or Egr‐1 deletion ameliorated endothelial cell migration toward VEGF‐A165 (vascular endothelial growth factor‐A) in a dual‐chamber model. On solubilized basement membrane preparations, Ser26 mutation or Egr‐1 deletion prevented endothelial network (or tubule) formation, an in vitro model of angiogenesis. Flow cytometry further revealed that Ser26 mutation or Egr‐1 deletion elevated early and late apoptosis. Finally, we demonstrated that Ser26 mutation or Egr‐1 deletion increased VE‐cadherin (vascular endothelial cadherin) expression, a regulator of endothelial adhesion and signaling, permeability, and angiogenesis.ConclusionsThese findings not only indicate that Egr‐1 is essential for endothelial cell proliferation, migration, and network formation, but also show that point mutation in Ser26 is sufficient to impair each of these processes and trigger apoptosis as effectively as the absence of Egr‐1. This highlights the importance of Ser26 in Egr‐1 for a range of proangiogenic processes.